Sulfamethoxazole molecular imprinting ratio fluorescent paper-based sensor based on rare earth complex fluorescent material as well as preparation method and application of sulfamethoxazole molecular imprinting ratio fluorescent paper-based sensor

Abstract

The invention discloses a sulfamethoxazole molecular imprinting ratio fluorescent paper-based sensor based on a rare earth complex fluorescent material as well as a preparation method and application of the sulfamethoxazole molecular imprinting ratio fluorescent paper-based sensor. The sulfamethoxazole molecularly imprinted ratiometric fluorescent paper-based sensor is prepared by the following steps: carrying out polymerization reaction on a red fluorescent material BCP-Eu, a green fluorescent material BCP-Tb, a template molecule sulfamethoxazole, a functional monomer and a cross-linking agent APTES on the surface of a paper base, eluting to remove the sulfamethoxazole template molecule, and detecting the sulfamethoxazole molecularly imprinted ratiometric fluorescent paper-based sensor. Finally, the sulfamethoxazole molecular imprinting ratio fluorescent paper-based sensor is obtained. According to the paper-based sensor, a molecular imprinting technology and a ratio fluorescence analysis method are organically combined, the paper-based sensor has the characteristics of high selectivity and high sensitivity, and specific recognition, rapid quantitative analysis and visual detection of sulfamethoxazole can be realized by further combining convenient imaging and signal reading functions of a smart phone; and an effective solution is provided for on-site instant detection of food safety.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of food safety visual detection technology, and particularly relates to a sulfamethoxazole molecular imprinting ratio fluorescent paper-based sensor based on a rare earth complex fluorescent material and a preparation method and application thereof. BACKGROUND

[0002] With the animal-derived food from demand-oriented to quality-oriented, the veterinary drug residue problem in animal-derived food has gradually become a global focus. Veterinary drug residue refers to the accumulation of original drugs, metabolites and impurities in the body of edible animals after the use of veterinary drugs or excretion through milk, eggs and other means. Veterinary drugs can be used in the prevention and treatment of animal diseases, and the improvement of livestock product quality. Improper use of drugs is the main reason for drug residues: (1) illegal use of prohibited drugs; (2) not following the drug-free period of veterinary drugs; (3) drug abuse, such as long-term (excessive) use of drugs and abuse of new or high-efficiency antibiotics; (4) use of drugs before slaughter. Due to the lack of scientific literacy of livestock breeders and the excessive pursuit of economic benefits, the irrational use of veterinary drugs is widespread in the current livestock industry. The phenomenon of drug abuse not only poses a direct threat to public health, but also greatly affects the sustainable development of the livestock industry and has serious adverse effects on the ecological environment.

[0003] Sulfonamides are widely used in the livestock industry. The antibacterial mechanism of sulfamethoxazole (SMZ) is to inhibit the synthesis of folic acid and interfere with the growth and reproduction of bacteria, leading to their death. This mechanism makes sulfamethoxazole play an important role in preventing and treating animal infectious diseases. Therefore, the food safety problem caused by its veterinary drug residue has attracted much attention. As a drug with significant antibacterial activity, sulfamethoxazole exhibits strong bactericidal efficacy against a variety of bacteria and performs well in preventing and treating animal bacterial infections. In addition, this drug also has the effect of reducing inflammation in the body, which is helpful for the repair and recovery process of the inflammation area, so it has been widely used in the pig industry. However, long-term intake of animal-derived food with excessive sulfonamide residues (cumulative amount) may affect the hematopoietic function, urinary system, and liver damage , induce allergic reactions, and even pose a potential risk of carcinogenesis. Improper use of sulfonamides has caused veterinary drug residue problems, which has become an important factor of environmental pollution and a threat to human health, and this problem needs to be effectively solved. At present, the methods for determining sulfamethoxazole mainly include mass spectrometry, high-performance liquid chromatography, liquid chromatography-mass spectrometry, solid-phase extraction-tandem mass spectrometry, enzyme-linked immunoassay, etc. Although these methods can accurately detect the concentration of sulfamethoxazole, they are expensive, have high detection costs, and are complex and time-consuming in pretreatment, making it difficult to analyze on site quickly. Therefore, it is of great practical significance to develop a low-cost, rapid, visual and portable visual detection method.

[0004] Paper-based visual rapid detection technology plays an increasingly significant role in food safety supervision. Its technical core advantage is reflected in the rapid and simple on-site detection of public health and emergency incidents. Ratio fluorescent probe, as a cutting-edge detection method, can obtain key signal parameters by comparing and analyzing the intensity ratio of fluorescent signals at two different wavelengths. This technology is particularly suitable for paper-based visual rapid detection applications. Ratio fluorescent probe can effectively eliminate external interference factors (such as light source fluctuation and equipment sensitivity difference) through dual-wavelength signal ratio correction mechanism. Its detection sensitivity and accuracy are significantly better than traditional single-signal probes, and data repeatability is better. Due to the complexity and diversity of food samples, ratio fluorescent paper-based visual rapid detection often shows low sensitivity and accuracy. Literature shows that molecular imprinting technology has unique advantages in solving the problem of large sample interference. Therefore, based on the good selectivity of molecular imprinting technology and the rapid and simple advantages of ratio fluorescent paper-based visual detection technology, this patent carries out research on the combination of molecular imprinting technology and ratio fluorescent paper-based sensor in food safety visual supervision and detection technology. This has important significance for realizing the accurate and rapid detection of trace harmful substances in animal-derived food samples.

[0005] This patent prepares two kinds of binuclear complex fluorescent materials BCP-Eu and BCP-Tb. Carboxylated solvent is used to modify the paper base, so that the paper base is rich in -COOH. The -COOH contained in the paper base can interact with the -NH2 in the template molecule SMZ through hydrogen bond. At the same time, based on the weakening effect of sulfamethoxazole (SMZ) on the green fluorescence emitted by BCP-Tb (“off”), and the enhancing effect of sulfamethoxazole (SMZ) on the red light emitted by BCP-Eu (“on”), a “dual-response-on-off” sulfamethoxazole molecular imprinting ratio fluorescent paper-based sensor (MIPs-SMZ@BCP-Tb / BCP-Eu@-COOH paper) is prepared in situ, which is used for detecting the residual concentration of SMZ in animal-derived food. SUMMARY

[0006] OBJECTIVE

[0007] Practice shows that there are still some problems to be solved in the molecular imprinting ratio fluorescent paper-based sensor in animal-derived food: (1) The detection effect of the ratio fluorescent probe depends on the selection of two kinds of fluorescent materials. Rare earth complexes are an important class of fluorescent materials, but as ratio fluorescent probe materials, there are still few molecular imprinting ratio fluorescent paper-based sensors prepared; (2) There are many types of paper-based materials, but there is a lack of paper-based materials suitable for the generation of molecular imprinting polymers; (3) How to combine the molecular imprinting polymer with the ratio fluorescent probe on the paper-based material to construct the molecular imprinting ratio fluorescent paper-based sensor, which is related to the popularization and application of the sensor in visual detection. The purpose of the patent application is to solve the above problems of the molecular imprinting ratio fluorescent paper-based sensor by the following methods: (1) Based on the fact that some rare earth complexes can emit fluorescence in the visible light range, which is an ideal visual fluorescent material, a series of pyridine carboxylic acid rare earth complexes are designed and prepared, and new rare earth complexes are obtained as ratio fluorescent probe materials, which lay a good foundation for the preparation of molecular imprinting ratio fluorescent paper-based sensors; (2) The paper-based material is modified to become an ideal carrier for the generation of molecular imprinting ratio fluorescent polymers; (3) Through the "one-step solution method", the molecular imprinting polymer and the ratio fluorescent probe are formed on the functionalized paper-based material at the same time, and a new method for detecting sulfamethoxazole (SMZ) residues in animal-derived food by a molecular imprinting ratio fluorescent paper-based sensor with double signal self-correction function is established. Combined with a smart phone, the on-site visual rapid quantitative detection of food safety is realized.

[0008] In order to achieve the above purpose, the application provides the following technical scheme:

[0009] A preparation method and analysis application of a sulfamethoxazole molecular imprinting ratio fluorescent paper-based sensor based on rare earth complex fluorescent materials, comprising the following steps:

[0010] (1) Preparation of carboxylated cellulose paper-based material: cut the 102 medium speed qualitative filter paper cellulose paper into 14 mm cellulose paper discs, put them into a culture dish, acidify with 20 mL of 0.2 M hydrochloric acid, avoid light and oscillate for 20 min, wash the cellulose paper discs with deionized water until the washing liquid is neutral, add 20 mL of 0.1 M ethylenediaminetetraacetic acid (EDTA) solution, avoid light and oscillate for 2.5 h, wash the cellulose paper discs with deionized water for 3-5 times, and put them into a 40℃ vacuum drying oven for drying to obtain a carboxylated cellulose paper-based material, which is marked as -COOH paper and ready for use;

[0011] (2) Preparation of the binuclear europium complex fluorescent material: 0.0493 g of [2,2'-bipyridine]-6,6'-dicarboxylic acid H2L and 0.0981 g of Eu(NO3)3·6H2O were weighed and dissolved in 6 mL of N,N-dimethylformamide (DMF) and 4 mL of H2O, and the uniform mixture was moved into a 25 mL reaction kettle lined with polytetrafluoroethylene after magnetic stirring until the solution was clear. The reaction was heated at 90°C in an oven for 72 h, and after the reaction was completed, the temperature was reduced to 50°C at a speed of 10°C / h, and maintained for 6 h. The gray powdery block crystals were obtained by filtration, washed with DMF, and dried at 60°C under vacuum for 12 h. Thus, the binuclear europium complex fluorescent material was obtained, which was recorded as BCP-Eu;

[0012] (3) Preparation of the binuclear terbium complex fluorescent material: 0.0336 g of [2,2'-bipyridine]-6,6'-dicarboxylic acid H2L and 0.0680 g of Tb(NO3)3·6H2O were weighed and dissolved in 6 mL of DMF and 4 mL of H2O, and the uniform mixture was moved into a 25 mL reaction kettle lined with polytetrafluoroethylene after magnetic stirring until the solution was clear. The reaction was heated at 90°C in an oven for 72 h, and after the reaction was completed, the temperature was reduced to 50°C at a speed of 10°C / h, and maintained for 6 h. The orange block crystals were obtained by filtration after standing until the crystals precipitated, washed with DMF, and dried at 60°C under vacuum for 12 h. Thus, the binuclear terbium complex fluorescent material was obtained, which was recorded as BCP-Tb.

[0013] (4) Development of a sulfamethoxazole molecularly imprinted ratio fluorescent paper-based sensor: 0.0100 g of the template molecule sulfamethoxazole (SMZ), 0.0050 g of the fluorescent material BCP-Eu obtained in step (2), 0.0100 g of the fluorescent material BCP-Tb obtained in step (3), 50.0 mg of a functional monomer and a crosslinking agent γ-aminopropyl triethoxysilane (APTES) were accurately weighed, 20 mL of anhydrous ethanol was measured in a conical flask, and after oscillation for 40 min, 0.5 mL of ammonia water was added and oscillation was continued for 20 min. The mixture was poured into a culture dish lined with the carboxylated cellulose paper obtained in step (1), and polymerization was carried out at 40°C in the dark for 9 h to allow sufficient reaction. After the reaction was completed, further elution was carried out with an eluent prepared from anhydrous methanol and acetic acid at a ratio of 9:1 until the eluent was free of SMZ. Vacuum drying (40°C) was carried out for 24 h, and thus the sulfamethoxazole molecularly imprinted ratio fluorescent paper-based sensor was obtained, which was recorded as MIPs-SMZ@BCP-Tb / BCP-Eu@-COOH paper.

[0014] (5) Preparation of a working curve

[0015] The fluorescence color was observed with a fixed excitation wavelength of 365 nm in a dark box ultraviolet analyzer, a smartphone (Xiaomi 10S) was used to take the fluorescence color of the paper-based sensor, and a color recognition APP was used to read the RGB value. A standard working curve was established with the logarithm (lgC SMZ ) of the SMZ concentration as the horizontal coordinate and the G' value as the vertical coordinate. The linear equation and correlation coefficient were obtained, and the LOD was calculated.

[0016] (6) Actual sample analysis

[0017] Preparation of pretreated sample solution: 8.0 g of chopped commercially available pork was accurately weighed and mixed with 20 mL of 0.1 M PBS solution. A magnetic stirrer was used to mix the meat sample and PBS solution thoroughly, and the mixture was then subjected to ultrasonic treatment for 45 min to ensure that the sample was completely dispersed in the solvent, which helped to effectively extract the target analyte from the pork matrix, thereby improving the quality and accuracy of the analysis. Then, the mixture was centrifuged at 4000 rpm for 15 min, and the clear solution containing the extracted analyte was collected, which was the pretreated sample solution.

[0018] Standard addition recovery experiment on the pretreated sample solution: sulfamethoxazole SMZ was added to the pretreated sample solution at concentrations of 0.1, 1.0, 3.0, 10.0 and 15.0 μM, respectively, and then the sulfamethoxazole molecularly imprinted ratio fluorescent paper-based sensor combined with a smartphone was used to visually determine and analyze the concentration of SMZ in the pork sample.

[0019] The biggest advantage and effect of the present invention mainly lies in:

[0020] (1) A series of rare earth complexes of [2,2'-bipyridine]-6,6'-dicarboxylic acid ligand were designed and prepared, and new rare earth complexes were obtained as ratio fluorescent probe materials;

[0021] (2) The paper-based material was carboxylated and modified to become an ideal carrier for the generation of molecularly imprinted ratio fluorescent polymers;

[0022] (3) Using the dual role of APTES "functional monomer + crosslinking agent", through the amino group (-NH2) at the end of the APTES molecule, on the one hand, it forms a specific complex with the template molecule SMZ, providing a basis for the formation of "imprinted" sites later, on the other hand, it can form a chemical bond with the carboxyl-containing paper base, anchoring the specific complex formed with the template molecule SMZ on the surface of the paper base; through the hydrolysis reaction of the triethoxysilane group (-Si(OEt)3) of APTES, silicon hydroxyl (-SiOH) is generated, based on which, on the one hand, the condensation reaction between different APTES can be carried out, constructing a crosslinked network structure, on the other hand, the silicon hydroxyl on APTES can condense with the hydroxyl of the cellulose in the paper base, further enhancing the binding force between APTES and the paper base;

[0023] (4) A preparation method of a BCP-Tb / BCP-Eu "dual response-switching" sulfonamidomethoxazole molecularly imprinted ratio fluorescent paper-based sensor based on pyridine carboxylic acid rare earth complex fluorescent materials is proposed, and when combined with a smart phone, visual rapid detection of sulfonamidomethoxazole residues in food is realized, which provides a possibility for visual rapid quantitative detection of food safety on site. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 (a) UV-Vis, Ex and Em spectra of BCP-Eu; (b) UV-Vis, Ex and Em spectra of BCP-Tb; (c) emission spectrum of BCP-Eu with an excitation wavelength of 325-335 nm; (d) emission spectrum of BCP-Tb with an excitation wavelength of 325-335 nm.

[0025] Figure 2 XRD patterns of paper, -COOH paper and MIPs-SMZ@BCP-Tb / BCP-Eu@-COOH paper.

[0026] Figure 3 SEM front view (a) and cross-section (b) of -COOH paper; SEM front view (c) and cross-section (d) of MIPs-SMZ@BCP-Tb / BCP-Eu@-COOH paper.

[0027] Figure 4 EDS element distribution map of -COOH paper.

[0028] Figure 5 EDS element distribution map of MIPs-SMZ@BCP-Tb / BCP-Eu@-COOH paper.

[0029] Figure 6XPS (a) and high resolution spectra of MIPs-SMZ@BCP-Tb / BCP-Eu@-COOH paper sensor: (b) C1s; (c) N1s; (d) O1s; (e) Si2p; (f) Tb3d; (g) Eu3d; (h) S2p.

[0030] Figure 7 Linear relationship between RGB values of MIPs-SMZ@BCP-Tb / BCP-Eu@-COOH paper and the logarithm of SMZ concentration: (a) 0.01 ~ 20.0 μM; (b) color change diagram.

[0031] Figure 8 Fluorescence color change diagram of MIPs-SMZ@BCP-Tb / BCP-Eu@-COOH paper ratio fluorescence paper sensor for detecting SMZ concentration in animal-derived food. DETAILED DESCRIPTION

[0032] The application will be described in detail below with reference to the accompanying drawings and examples.

[0033] Example 1 Visual determination of sulfamethoxazole residue in pork samples

[0034] (1) Preparation of carboxylated cellulose paper base material: 102 medium qualitative filter paper cellulose paper was cut into cellulose paper discs with a diameter of 14 mm, placed in a culture dish, acidified with 20 mL of 0.2 M hydrochloric acid, and shaken in the dark for 20 min. The cellulose paper discs were washed with deionized water until the washing liquid was neutral, 20 mL of 0.1 M ethylenediaminetetraacetic acid (EDTA) solution was added, and the mixture was shaken in the dark for 2.5 h. The cellulose paper discs were washed 4 times with deionized water, placed in a vacuum drying oven at 40°C, and dried to obtain carboxylated cellulose paper base material, which was labeled as -COOH paper and stored for later use.

[0035] XRD patterns of MIPs-SMZ@BCP-Tb / BCP-Eu@-COOH paper, BCP-Tb / BCP-Eu@-COOH paper, and -COOH paper are shown in the accompanying Figure 2 SEM images of MIPs-SMZ@BCP-Tb / BCP-Eu@-COOH paper, BCP-Tb / BCP-Eu@-COOH paper, and -COOH paper are shown in the accompanying Figure 3 (a, b); and EDS element distribution maps of MIPs-SMZ@BCP-Tb / BCP-Eu@-COOH paper, BCP-Tb / BCP-Eu@-COOH paper, and -COOH paper are shown in the accompanying Figure 4 .

[0036] (2) Preparation of the binuclear europium complex fluorescent material: 0.0493 g of [2,2'-bipyridine]-6,6'-dicarboxylic acid H2L and 0.0981 g of Eu(NO3)3·6H2O were weighed and dissolved in 6 mL of N,N-dimethylformamide DMF and 4 mL of distilled water H2O, and the uniform mixture was moved to a 25 mL reaction kettle lined with polytetrafluoroethylene after magnetic stirring until the solution was clear. The reaction was heated at 90°C in an oven for 72 h, and after the reaction was completed, the temperature was reduced to 50°C at a speed of 10°C / h, and maintained for 6 h. The gray block-shaped crystals were obtained by filtration, washed with DMF, and dried under vacuum at 60°C for 12 h. The binuclear europium complex fluorescent material was obtained, denoted as BCP-Eu.

[0037] The optical performance test results of the binuclear europium complex fluorescent material are shown in the following table: Figure 1 (a, c).

[0038] (3) Preparation of the binuclear terbium complex fluorescent material: 0.0336 g of [2,2'-bipyridine]-6,6'-dicarboxylic acid H2L and 0.0680 g of Tb(NO3)3·6H2O were weighed and dissolved in 6 mL of DMF and 4 mL of distilled water H2O, and the uniform mixture was moved to a 25 mL reaction kettle lined with polytetrafluoroethylene after magnetic stirring until the solution was clear. The reaction was heated at 90°C in an oven for 72 h, and after the reaction was completed, the temperature was reduced to 50°C at a speed of 10°C / h, and maintained for 6 h. The orange block-shaped crystals were obtained by filtration, washed with DMF, and dried under vacuum at 60°C for 12 h. The binuclear terbium complex fluorescent material was obtained, denoted as BCP-Tb.

[0039] The optical performance test results of the binuclear terbium complex fluorescent material are shown in the following table: Figure 1 (b, d).

[0040] (4) Preparation of sulfamethoxazole molecularly imprinted ratio fluorescent paper-based sensor: 0.0100 g of template molecule sulfamethoxazole SMZ, 0.0050 g of fluorescent material BCP-Eu obtained in step (2), 0.0100 g of fluorescent material BCP-Tb obtained in step (3), 50.0 mg of functional monomer and crosslinking agent γ-aminopropyl triethoxysilane APTES were accurately weighed in a conical flask containing 20 mL of anhydrous ethanol, 0.5 mL of ammonia water was added after oscillation for 40 min, and oscillation was continued for 20 min. Pour it into a petri dish full of carboxylated cellulose paper obtained in step (1), and polymerize at 40°C in the dark for 9 h to allow sufficient reaction. After the reaction is completed, further elution is carried out with an eluent prepared from anhydrous methanol: acetic acid = 9:1 until the eluent does not contain SMZ. Dry at 40°C under vacuum for 24 h to obtain the sulfamethoxazole molecularly imprinted ratio fluorescent paper-based sensor, which is denoted as MIPs-SMZ@BCP-Tb / BCP-Eu@-COOH paper. The XRD pattern, EDS element distribution map and XPS map of MIPs-SMZ@BCP-Tb / BCP-Eu@-COOH paper are shown in Figs. 1, 2 and 3, respectively. Figure 2 , Fig. Figure 5 and Fig. Figure 6 .

[0041] (5) Preparation of working curve

[0042] Different concentrations (0.01 ~ 20.0 μM) of SMZ ethanol solution were added to the surface of the sulfamethoxazole molecularly imprinted ratio fluorescent paper-based sensor (MIPs-SMZ@BCP-Tb / BCP-Eu@-COOH paper) and completely wetted, and after drying at room temperature, the fluorescence color was observed on the dark box ultraviolet analyzer with a fixed excitation wavelength of 365 nm. The fluorescence color of the paper-based sensor was photographed using a smart phone (Xiaomi 10S), and the RGB value was read using a color recognition APP. A standard working curve was established with the logarithm (lgC SMZ ) of SMZ concentration as the abscissa and the Gʹ value as the ordinate. The linear equation and correlation coefficient were obtained, and the LOD was calculated.

[0043] The working curve is shown in Fig. Figure 7 , and the fluorescence color change diagram is shown in Fig. Figure 8 .

[0044] (6) Analysis of actual samples

[0045] Preparation of pretreated sample solution: 8.0 g of chopped pork was accurately weighed and mixed with 20 mL of 0.1 M PBS solution. The meat sample and PBS solution were mixed well using a magnetic stirrer, and the mixture was then subjected to ultrasonic treatment for 45 min. The mixture was then centrifuged at 4000 rpm for 15 min, and the clear solution containing the extracted analyte was collected, which was the pretreated sample solution.

[0046] Recovery experiment of the pretreated sample solution: Sulfamethoxazole (SMZ) was added to the pretreated sample solution at concentrations of 0.1, 1.0, 3.0, 10.0, and 15.0 μM, respectively. Subsequently, the concentration of SMZ in the pork sample was visually determined using the sulfamethoxazole molecularly imprinted ratio fluorescent paper-based sensor combined with a smartphone. The results of the recovery experiment are shown in Table 1 below.

[0047] Table 1 Visual detection of SMZ in animal-derived food using a ratio fluorescent paper-based sensor (n = 3)

[0048]

[0049] As shown in Table 1, the recovery rate of the MIPs-SMZ@BCP-Tb / BCP-Eu@-COOH paper ratio fluorescent paper-based sensor for detecting the concentration of SMZ in the pork pretreated sample solution was between 94.30 and 101.06%, with an RSD of less than 3.02%. These results demonstrate the successful application of fluorescent visualization in detecting the concentration of SMZ residues in animal-derived food.

[0050] Appendix Figure 1UV-Vis, Ex and Em spectra of BCP-Eu (a); UV-Vis, Ex and Em spectra of BCP-Tb (b); emission spectrum of BCP-Eu with excitation wavelength of 325~335 nm (c); emission spectrum of BCP-Tb with excitation wavelength of 325~335 nm (d). From the UV-Vis spectra of (a) and (b), the absorption peaks of BCP-Eu and BCP-Tb at 278 nm and 266 nm correspond to the unsaturated bond π→π* transition, which belongs to the B absorption band. While the absorption peaks at 320 nm and 303 nm belong to the R absorption band of the band lone pair electron chromophoric group (C=O) n→π* transition. In addition, in order to ensure that the fluorescence intensity of BCP-Eu and BCP-Tb reaches the strongest at the same time, the excitation wavelength in the range of 325~335 nm is explored. When the excitation wavelength is 328 nm, the emission spectrum of BCP-Eu and BCP-Tb has the strongest fluorescence intensity (in the figures (c) and (d)), and 328 nm is selected as the excitation wavelength for subsequent fluorescence analysis.

[0051] Figure 1 Figure 2 Figure 2 XRD patterns of paper, -COOH paper and MIPs-SMZ@BCP-Tb / BCP-Eu@-COOH paper. From the XRD pattern, it can be clearly seen that the XRD of the carboxylated paper-based material (-COOH paper) has a displacement compared with the original paper-based material (paper), which proves the success of the carboxylation of the paper-based material. However, compared with the characteristic peak of the -COOH paper, the characteristic diffraction peak of the MIPs-SMZ@BCP-Tb / BCP-Eu@-COOH paper-based sensor does not change in position, only the peak intensity is weakened. The unchanged characteristic diffraction peak position indicates that there is still -COOH on the paper-based material after polymerization, and the weakened diffraction peak intensity indicates that part of the -COOH on the paper-based material forms a hydrogen bond with the -NH2 in the template molecule SMZ, and participates in the imprinting polymerization reaction under the action of the crosslinking agent APTES, which also indirectly proves the successful preparation of the MIPs-SMZ@BCP-Tb / BCP-Eu@-COOH paper-based sensor.

[0052] Figure 3 Figure 3 (a, b) are SEM images of the front and cross section of the -COOH paper, from which the fiber bundles on the surface of the paper-based material can be clearly observed; Figure 4 Figure 3(c, d) are SEM images of the front and cross-section of the MIPs-SMZ@BCP-Tb / BCP-Eu@-COOH paper of the present application. The paper substrate surface is attached with a lot of reticular material, indicating that the molecular imprinting ratio fluorescent polymer material has been successfully grafted to the surface of the cellulose paper.

[0053] Figure 2 shows the EDS element distribution of the -COOH paper of the present application. Figure 4 (a) - (d) are EDS element distribution maps of the -COOH paper of the present application. It can be seen from the figure that the front of the paper substrate contains C, N and O elements, which confirms the success of the carboxylation of the paper substrate.

[0054] Figure 3 shows the XPS spectra of the MIPs-SMZ@BCP-Tb / BCP-Eu@-COOH paper of the present application. Figure 5 (a) - (g) are EDS element distribution maps of the MIPs-SMZ@BCP-Tb / BCP-Eu@-COOH paper of the present application. It can be seen from the figure that there are C, O, N, Si, Tb and Eu elements, which can confirm the success of the preparation of the molecular imprinting ratio fluorescent polymer.

[0055] Figure 4 shows the XPS spectra of the MIPs-SMZ@BCP-Tb / BCP-Eu@-COOH paper of the present application. Figure 6 (a) is the XPS spectrum of the MIPs-SMZ@BCP-Tb / BCP-Eu@-COOH paper substrate sensor of the present application. The results show that the prepared molecular imprinting ratio fluorescent paper substrate sensor mainly contains C, N, O, Si, Tb, Eu and S elements. In the attached Figure 6 (b), the high-resolution C1s spectrum of the MIPs-SMZ@BCP-Tb / BCP-Eu@-COOH paper shows four different carbon states at 284.80 eV, 286.26 eV, 288.16 eV and 290.86 eV, which correspond to C=C, C-O-C, O-C=O and π–π* satellite peaks, respectively. In the attached Figure 6 (c), the N1s spectrum shows three peaks at 397.99 eV, 399.47 eV and 406.06 eV, which correspond to C-N=C, N-H and π–π* satellite peaks. In the attached (d), the O1s spectrum shows peaks at 531.14 eV, 532.12 eV and 533.17 eV, which may correspond to RE-O, C-O and C=O. The Si2p doublet of the MIPs-SMZ@BCP-Tb / BCP-Eu@-COOH paper ratio fluorescent paper substrate sensor has a binding energy of 103.18 eV and 102.08 eV, which is attributed to Si2p1and Si2p3(attachment Figure 6 (e)). In the attached Figure 6(f) In (f), the high-resolution Tb3d spectrum of the paper-based sensor shows peaks at 1276.61 eV, 1241.61 eV and 1225.26 eV, which are attributed to Tb3d3, Tb3d5, shake-down satellite peaks, respectively. While the Figure 6 (g) In (g), the Eu3d spectrum shows peaks at 1135.09 eV, 1165.09 eV and 1123.70 eV, which are attributed to Eu3d3, Eu3d5, shake-down satellite peaks, respectively. In addition, the S2p spectrum of trace residual template molecule SMZ in the eluted MIPs-SMZ@BCP-Tb / BCP-Eu@-COOH paper ratio fluorescent paper-based sensor has two double peaks with binding energies of 168.46 eV, 169.63 eV and 163.98 eV, 164.80 eV, which are related to S=O S2p3, S=O S2p1 and C-S S2p3, C-S S2p1 (see Figure 6 (h) in the accompanying drawings).

[0056] The standard working curve and linear relationship for visualizing the detection of SMZ concentration by the sulfamethoxazole molecularly imprinted ratio fluorescent paper-based sensor (MIPs-SMZ@BCP-Tb / BCP-Eu@-COOH paper) are shown in Figure 7 The accompanying drawings show that Figure 7 (a) shows that in the range of 0.01 ~ 1.5 μM, the Gʹ value of RGB has a linear relationship with the logarithm of SMZ concentration (lgC SMZ ), and the linear equation is y1 = -20.8368 x1+ 213.4078 (R 2 = 0.9962); in the range of 1.5 ~ 10.0 μM, the linear equation is y2 = -47.6958 x2+ 211.9553 (R 2 = 0.9948); in the range of 10.0 ~ 20.0 μM, the linear equation is y3= -109.6711 x3+ 269.4247 (R 2 = 0.9954); and the LOD for detecting the concentration of SMZ is 67.9 nM. As shown in Figure 7 (b), the color change of the MIPs-SMZ@BCP-Tb / BCP-Eu@-COOH paper-based sensor is: green → yellow → orange → orange red.

[0057] To further explore the practicality of MIPs-SMZ@BCP-Tb / BCP-Eu@-COOH paper, the standard addition method was used to add SMZ standard solution (concentrations were 0.1 μM, 1.0 μM, 3.0 μM, 10.0 μM and 15.0 μM) in the pretreated sample solution of pork, and the results of color change of paper-based sensor were shown in Fig. 6. Figure 8 The results of color change of paper-based sensor were shown in Fig. 6.

Claims

1. A method for preparing a sulfamethoxazole molecularly imprinted ratiometric fluorescent paper-based sensor based on rare-earth complex fluorescent materials, characterized in that, Includes the following steps: (1) Preparation of carboxylated cellulose paper base material: Cut cellulose paper of medium speed qualitative filter paper of 102 into cellulose paper discs with a diameter of 14 mm, place them in a petri dish, acidify with 20 mL of 0.2 M hydrochloric acid, shake in the dark for 20 min, wash the cellulose paper discs with deionized water until the washing solution is neutral, add 20 mL of 0.1 M EDTA solution, shake in the dark for 2.5 h, wash the cellulose paper discs with deionized water 3-5 times, place them in a vacuum drying oven at 40℃, and dry to obtain carboxylated cellulose paper base material, denoted as -COOH paper, for later use; (2) Preparation of binuclear europium complex fluorescent material: Weigh 0.0493 g of [2,2′-bipyridine]-6,6′-dicarboxylic acid H2L and 0.0981 g of Eu(NO3)3·6H2O, dissolve them in 6 mL of N,N-dimethylformamide DMF and 4 mL of distilled water H2O, stir magnetically until the solution is clear, transfer the homogeneous mixture to a 25 mL polytetrafluoroethylene-lined reactor, heat it in an oven at 90℃ for 72 h, after the reaction is complete, reduce the temperature to 50℃ at a rate of 10℃ / h, maintain for 6 h, filter to obtain grayish-pink block crystals, wash with DMF, dry under vacuum at 60℃ for 12 h to obtain binuclear europium complex fluorescent material, denoted as BCP-Eu; (3) Preparation of binuclear terbium complex fluorescent material: Weigh 0.0336 g of [2,2′-bipyridine]-6,6′-dicarboxylic acid H2L and 0.0680 g of Tb(NO3)3·6H2O and dissolve them in 6 mL of DMF and 4 mL of distilled water H2O. Stir magnetically until the solution is clear. Transfer the uniformly mixed solution to a 25 mL reaction vessel lined with polytetrafluoroethylene. Heat the reaction at 90 °C in an oven for 72 h. After the reaction is complete, reduce the temperature to 50 °C at a rate of 10 °C / h and keep it for 6 h. Let it stand until crystals precipitate. Filter to obtain orange block crystals. Wash with DMF and dry under vacuum at 60 °C for 12 h to obtain the binuclear terbium complex fluorescent material, denoted as BCP-Tb. (4) Development of a sulfamethoxazole molecularly imprinted ratio fluorescent paper-based sensor: Accurately weigh 0.0100 g of template molecule sulfamethoxazole SMZ, 0.0050 g of fluorescent material BCP-Eu obtained in step (2), 0.0100 g of fluorescent material BCP-Tb obtained in step (3), 50.0 mg of functional monomer and crosslinking agent γ-aminopropyltriethoxysilane APTES, measure 20 mL of anhydrous ethanol into a conical flask, shake for 40 min, add 0.5 mL of ammonia water, continue shaking for 20 min, pour it into a petri dish covered with carboxylated cellulose paper base obtained in step (1), and polymerize at 40℃ in the dark for 9 h to allow it to react fully. After the reaction is complete, elute further with an eluent prepared with anhydrous methanol: acetic acid = 9:1 until the eluent does not contain SMZ, and dry in a vacuum drying oven at 40℃ for 24 hours. h, thus obtaining the sulfamethoxazole molecular imprint ratio fluorescent paper-based sensor, denoted as MIPs-SMZ@BCP-Tb / BCP-Eu@-COOH paper.

2. The molecularly imprinted ratiometric fluorescent paper-based sensor prepared according to claim 1 can be used for the visual detection of sulfamethoxazole residues in meat products, characterized in that, The steps include the following: The sample solution to be tested was added dropwise to the sulfamethoxazole molecularly imprinted ratio fluorescent paper-based sensor MIPs-SMZ@BCP-Tb / BCP-Eu@-COOH paper prepared by the method described in claim 1, allowed to air dry naturally, and observed under a 365 nm ultraviolet lamp. The RGB values ​​were obtained using smartphone color analysis software for visual detection and analysis.

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